DESCRIPTION(adapted from applicant's abstract): There is growing evidence that ROS may be implicated in aging and a variety of age-related human degenerative disorders. In this context, exogenous antioxidants are beginning to assume an unprecedented importance in the arsenal of therapeutic agents that are continuously being developed in order to combat age related diseases and the phenomenon of aging itself. These antioxidants are supposed to supplement and reinforce the natural defenses of the body and to protect it when the endogenous antioxidant levels are diminished as a result of disease or aging. The current problems in the in vitro assay of antioxidants are many and varied: (a) different laboratories use different species of ROS and different target molecules under different experimental conditions in formulating their assay systems; (b) there are no comparative studies to evaluate the reliability of one method over an other, (c) many have been mainly employed for investigating the mechanisms of action of specific oxygen free radicals and are not particularly relevant as procedures for the assay of antioxidants; (d) since many of the methods are not specifically designed for antioxidant assay, they have not been optimized taking into consideration the pro-oxidant or antioxidant properties of certain substances under different conditions; and (e) they often require expensive instrumentation and highly sophisticated technical skill to perform the analysis and to interpret the resulting data. In the first step in this project, our goal will be to develop in vitro assay systems for the measurement of individual biological activities of coenzyme Q, lipoic acid and melatonin, using pure commercial preparations of these compounds, taking into consideration the properties of both the ROS and the antioxidants involved. In the second step, we will develop an in vivo method using for determining antioxidant activity using intact cells and intracellular macromolecules as targets. The results of the two systems will be analyzed to yield an approach which can be employed for the determination of biological activities of known or unknown mixtures of antioxidants using a minimum number of simple assay procedures.